PROJECT SUMMARY: PROJECT 3 There is a continuing need to develop non-vaccine biomedical prevention (nBP) strategies against sexual HIV transmission that can be delivered topically to the vaginal mucosa. Next generation nBP candidates likely will include sustained release delivery platforms eluting multiple antiretroviral (ARV) agents, but efficacy evaluation in such platforms is multifactorial and difficult to assess accurately. Traditional ex vivo systems for ARV efficacy screening are limited by low throughput due to: high cost and limited availability of cultures and animals; variability of culture quality; the underestimated impact of genetics and unknown donor pathologies; and the frequent ectocervical, rather than vaginal, origin of the tissues. To meet this important need, we have developed a novel, high-throughput, reproducible vaginal epithelial cell (VEC) culture system that develops into multilayers and can be co-cultured with HIV-susceptible immune cells. The system recreates many of the anatomic and physiologic characteristics of the vaginal mucosa, including intact commensal vaginal bacterial community colonization and productive HIV transmission and infection. The overarching goal of Project 3 is the identification of the most efficacious multi-ARV combination for intravaginal ring (IVR) formulation to prevent sexual HIV transmission, starting with a library of nine candidates formulated under Core B. PK profiles from these candidates IVRs tested in sheep and mice (Project 1) will provide target drug concentrations for efficacy testing, and will direct multi-drug formulation selection for subsequent pre-clinical, IND-enabling activities (Projects 1, 2 & 5). In Aim 1, the nine candidate ARV formulations will be screened for efficacy against HIV using our multilayer VEC-monocyte-derived macrophage (VEC-MDM) culture model. The co-cultures will be colonized with complex vaginal microbiota representing the main community types including dysbiotic states. Vaginal fluids from women treated with our lead candidate ARV IVR (Project 4) will be assessed to confirm surrogate efficacy ex vivo. The nine candidate ARV IVRs will be studied in parallel for efficacy in preventing HIV infection in BLT humanized mice (Aim 2), and these activities will be highly interrelated with those carried out in Aim 1. In Aim 3, the best performing candidate ARV IVR, selected primarily based on results from Aims 1 and 2 as well as safety outcomes (Project 2), will be will be evaluated in the macaque SHIV repeat challenge model to confirm the nBP candidate's ability to provide complete protection from intravaginal infection in preparation for future clinical studies.